scholarly journals The roles of APE1, APE2, DNA polymerase β and mismatch repair in creating S region DNA breaks during antibody class switch

2008 ◽  
Vol 364 (1517) ◽  
pp. 645-652 ◽  
Author(s):  
Carol E Schrader ◽  
Jeroen E.J Guikema ◽  
Xiaoming Wu ◽  
Janet Stavnezer
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Cytidine Deaminase ◽  
Dna Breaks ◽  
Dna Polymerase Β ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Class Switch ◽  
Single Strand Breaks ◽  
Antibody Class

Immunoglobulin class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded DNA breaks (DSBs) in immunoglobulin switch region DNA. The initial steps of DSB formation have been elucidated: cytosine deamination by activation-induced cytidine deaminase (AID) and the generation of abasic sites by uracil-DNA glycosylase (UNG). We show that abasic sites are converted into single-strand breaks (SSBs) by apurinic/apyrimidinic endonucleases (APE1 and APE2). If SSBs are near to each other on opposite strands, they will generate DSBs; but if distal from each other, mismatch repair appears to be required to generate DSBs. The resulting S region DSBs occur at dC residues that are preferentially targeted by AID. We also investigate whether DNA polymerase β, which correctly repairs SSBs resulting from APE activity, attempts to repair the breaks during CSR. We find that although polymerase β does attempt to repair S region DNA breaks in switching B cells, the frequency of AID-instigated breaks appears to outnumber the SSBs repaired correctly by polymerase β, and thus some DSBs and mutations are generated. We also show that the S region DSBs are introduced and resolved during the G1 phase of the cell cycle.

scholarly journals APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination

2007 ◽  
Vol 204 (12) ◽  
pp. 3017-3026 ◽  
Author(s):  
Jeroen E.J. Guikema ◽  
Erin K. Linehan ◽  
Daisuke Tsuchimoto ◽  
Yusaku Nakabeppu ◽  
Phyllis R. Strauss ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Uracil Dna Glycosylase ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Antibody Class

Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2.

scholarly journals DNA polymerase β is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination

2007 ◽  
Vol 204 (7) ◽  
pp. 1677-1689 ◽  
Author(s):  
Xiaoming Wu ◽  
Janet Stavnezer
Keyword(s):  
B Cells ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Strand Break ◽  
Dna Polymerase Β ◽  
Single Strand Break ◽  
Class Switch ◽  
Base Excision

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase β (Polβ) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polβ might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polβ levels are induced upon activation of splenic B cells to undergo CSR. When Polβ−/− B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polβ attempts to faithfully repair S region lesions but fails to repair them all.

scholarly journals DNA Polymerase η Is Involved in Hypermutation Occurring during Immunoglobulin Class Switch Recombination

2004 ◽  
Vol 199 (2) ◽  
pp. 265-270 ◽  
Author(s):  
Ahmad Faili ◽  
Said Aoufouchi ◽  
Sandra Weller ◽  
Françoise Vuillier ◽  
Anne Stary ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Tandem Repeats ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Class Switch ◽  
Mutation Pattern ◽  
Immunoglobulin Locus ◽  
V Gene

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase η (pol η), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol η is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.

scholarly journals Regulation of class switch recombination and somatic mutation by AID phosphorylation

2008 ◽  
Vol 205 (11) ◽  
pp. 2585-2594 ◽  
Author(s):  
Kevin M. McBride ◽  
Anna Gazumyan ◽  
Eileen M. Woo ◽  
Tanja A. Schwickert ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Dna Breaks ◽  
Class Switching ◽  
Class Switch

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

scholarly journals The cohesin complex regulates immunoglobulin class switch recombination

2013 ◽  
Vol 210 (12) ◽  
pp. 2495-2502 ◽  
Author(s):  
Anne-Sophie Thomas-Claudepierre ◽  
Ebe Schiavo ◽  
Vincent Heyer ◽  
Marjorie Fournier ◽  
Adeline Page ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Cohesin Complex ◽  
Dna Breaks ◽  
End Joining ◽  
Regulatory Subunits ◽  
Class Switch ◽  
Chromatid Cohesion ◽  
Switch Regions

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the Sμ-Cμ region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining.

scholarly journals Origin of Endogenous DNA Abasic Sites in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (22) ◽  
pp. 8386-8394 ◽  
Author(s):  
Marie Guillet ◽  
Serge Boiteux
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Delay ◽  
Dna Glycosylases ◽  
Triple Mutant ◽  
Gene Encoding ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Single Strand Breaks ◽  
Genes Encoding ◽  
Ap Sites

ABSTRACT Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3′-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G2/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.

scholarly journals Inducible DNA breaks in Ig S regions are dependent on AID and UNG

2005 ◽  
Vol 202 (4) ◽  
pp. 561-568 ◽  
Author(s):  
Carol E. Schrader ◽  
Erin K. Linehan ◽  
Sofia N. Mochegova ◽  
Robert T. Woodland ◽  
Janet Stavnezer
Keyword(s):  
Cytidine Deaminase ◽  
Constant Region ◽  
Dna Breaks ◽  
Region Gene ◽  
Uracil Dna Glycosylase ◽  
Strand Breaks ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Constant Region Gene ◽  
Dna Strands

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cμ) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.

scholarly journals Immunoglobulin Class Switch Recombination Is Initiated by Rare Cytosine Deamination Events at Switch Regions

2020 ◽  
Vol 40 (16) ◽  
Author(s):  
Ahrom Kim ◽  
Li Han ◽  
Kefei Yu
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Strand Break ◽  
Switch Region ◽  
Cytosine Deamination ◽  
Class Switch ◽  
Switch Regions ◽  
Dna Strands ◽  
Uracil Repair

ABSTRACT Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.

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